Flue-cured tobacco production in China is typically over-fertilized and mono-cropped. To understand how this agronomic management affects soil structure and organic matter, this study investigated the effect of rotation, fertilizer rate and manure amendment on the proportion of water stable aggregates and aggregate-associated soil organic carbon (SOC) and total soil nitrogen (TSN) concentrations and stocks in tobacco production. Two tobacco rotation systems (tobacco monocropping and tobacco-rice) with four fertilizer managements (0, 75, and 112 kg N/ha, and 60 kg N/ha + Manure) were established in 1998. Eighteen years after treatments were implemented, soil aggregation, and aggregate-associated SOC and TSN were significantly affected by rotation and fertilizer management. Compared to tobacco monoculture and current fertilizer management, rotation and manure amendment increased macroaggregate (>250 µm) proportion and geometric mean diameter, but decreased the proportion of microaggregates and silt-clay sized fractions (<250 µm). Rotation and manure amendment simultaneously increased the percentage of macroaggregate fractions and associated SOC and TSN stocks at the expense of the microaggregate and silt-clay size class and associated SOC and TSN stocks. Using rotation and/or manure amendment in tobacco production appears to maintain desirable soil physical and chemical properties via macroaggregates stabilization, which leads to the conservation of SOC and TSN stocks.